Analysis of porcine circovirus type 1 detected in Rotarix vaccine
Analysis of porcine circovirus type 1 detected in Rotarix vaccine
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DOI：
10.1016/j.vaccine.2010.11.028
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发表时间：
2011-01-17
期刊：
影响因子：
5.5
通讯作者：
Mankertz, Annette
中科院分区：
文献类型：
--
作者：
Baylis, Sally A.;Finsterbusch, Tim;Mankertz, Annette
最近对人活疫苗进行的宏基因组分析表明，用于预防急性胃肠炎的小儿疫苗Rotarix中存在猪圆环病毒1型（PCV1） DNA。对PCV1进行实时PCR检测，发现几批Rotarix中PCV1 DNA的滴度为每剂量6-7 log（10）份。用核酸酶苯并酶预处理重组疫苗，然后提取核酸并通过实时PCR定量检测PCV1 DNA，结果表明与未处理的对照组相比，重组疫苗没有丢失PCV1 DNA滴度，这表明猪病毒DNA以封装形式存在于疫苗中。分别用Rotarix或PCV1感染PCV1允许的PS细胞、人HEK293和Vero细胞，并进行免疫荧光和RT-PCR检测。病毒基因组存在于rotarix孵育和pcv1感染的细胞中，而病毒转录仅在pcv1感染的细胞中可见。同样，在pcv1感染的细胞中观察到pcv1特异性蛋白的表达，但在Rotarix处理的细胞中没有。上清传代表明，在pcv1感染的PS细胞中产生感染，但在HEK293和Vero细胞或与Rotarix孵育的任何细胞系中没有产生感染。存在于轮虫体内的PCV1 DNA不受苯并酶的酶切；然而，PCV1在免疫电镜下未被识别，无法感染PS、HEK293或Vero细胞，这表明在Rotarix中存在大量PCV1 DNA并不反映出相应比例的生物活性病毒颗粒。(C) 2010 Elsevier Ltd.版权所有。
A metagenomic analysis of live human vaccines has recently demonstrated the presence of porcine circovirus type 1 (PCV1) DNA in the paediatric vaccine Rotarix used in the prevention of acute gastroenteritis. Using real-time PCR for PCV1, titres of PCV1 DNA in several batches of Rotarix were found to be in the order of 6-7 log(10) copies per dose. Pre-treatment of the reconstituted vaccine with the nuclease Benzonase, followed by extraction of nucleic acid and quantification of PCV1 DNA by real-time PCR, revealed that there was no loss of PCV1 DNA titre compared to untreated controls, suggesting that the porcine viral DNA was present in the vaccine in an encapsidated form. PCV1 permissive PS cells, human HEK293 and Vero cells, used for vaccine production, were infected with Rotarix or PCV1, respectively, and subjected to immune fluorescence and RT-PCR. Viral genomes were present in Rotarix-incubated as well as PCV1-infected cells, while viral transcription was seen only in PCV1-infected cells. Similarly, PCV1-specific protein expression was observed in PCV1-infected cells, but not in cells treated with Rotarix. Passaging of the supernatant indicated productive infection in PCV1-infected PS cells, but not in HEK293 and Vero cells or in any cell line incubated with Rotarix. PCV1 DNA present in Rotarix was protected from Benzonase digestion; however, PCV1 was not recognized in immune electron microscopy and unable to infect PS, HEK293 or Vero cells, suggesting that the high amount of PCV1 DNA present in Rotarix does not reflect a corresponding proportion of biologically active virus particles. (C) 2010 Elsevier Ltd. All rights reserved.